[unreadable] NIDDK's mission includes research in urologic and digestive diseases to improve the health of Americans. Recent studies indicate that organs, such as the urinary bladder and stomach, may contain stem cells capable of regenerating damaged tissue. This award will train Dr. Monica Liebert, a senior investigator in urothelial (urinary bladder) biology, in stem cell techniques. Liebert will be co-mentored by Max Wicha, leader in breast cancer stem cell biology research, and Deborah Gumucio, a expert in gastrointestinal organogenesis. Wicha will train Liebert in basic stem cell isolation, characterization and culture techniques. Liebert will then apply that training to study gastric stem cells. Gumucio previously found that transgenes driven by the villin promoter are expressed at high levels in a subset of gastric cells located in tissue where gastric stem cells are thought to reside. Our hypothesis is that these cells are a subset of a larger gastric stem cell population. Aim 1 is to isolate and characterize stem-like gastric stem cells. Cells with stem cell characteristics will be enriched from tissues using magnetic or flow cytometric separation. These cells will be cultured to form anchorage-independent "gastrospheres," and evaluated for stem cell properties and gene expression. Antibody staining, microarray methods and real-time quantitative PCR will be used to examine the these cells and compare them to the cells identified by villin-promoter driven EGFP expression. Aim 2 will test whether these stem-like cells can give rise to intestinal or gastric lineages in vitro or in vivo. Cells will be cultured under differentiating conditions. Enriched populations of gastric stem cells from Rosa-26 mice and the Villin-EGFP x Rosa 26 cross, as well as a control population of unsorted cells, will be isolated and grafted on nude mice. Xenografts will be evaluated for EGFP, beta-gal expression and for morphologic development into intestinal or gastric tissues. These studies will confirm the ability of the different potential stem cell populations to reproduce and to differentiate. This research will help us understand how the stomach responds to diseases, such as ulcers, and how stomach cancer may develop. At the end of Liebert's training, she will apply these techniques to the study of urinary bladder development, tissue engineering, and bladder cancer. Her future work in urology will develop new treatments for urinary tract disorders and bladder cancer. [unreadable] [unreadable] [unreadable]